1. Field of the Invention
This invention relates to a method for rapidly detecting the presence of duplex formation between single stranded nucleotide macromolecules, and more specifically, this invention relates to a method for using oligonucleotide arrays to rapidly detect duplex formation of oligonucleotide sequences. This invention also relates to a simple procedure for producing the oligonucleotide-arrays.
2. Background of the Invention
Present techniques for determining the existence of target sequences in patient DNA are complex, inefficient and somewhat time consuming. For example, one multistep DNA sequencing approach, the Maxam and Gilbert method, involves first labeling DNA, and then splitting the DNA with a chemical, designed to alter a specific base, to produce a set of labeled fragments. The process is repeated by cleaving additional DNA with other chemicals specific for altering different bases, to produce additional sets of labeled fragments. The multiple fragment sets then must be run side-by-side in electrophoresis gels to determine base sequences.
Another sequencing method, the dideoxy procedure, based on Sanger, et al. Proc. Natl. Acad. Sci. USA 74, 5463-7 (1977) first requires the combination of a chain terminator as a limiting reagent, and then the use of polymerase to generate various length molecules, said molecules later to be compared on a gel. The accompanying lengthy electrophoresis procedures further detracts from the utility of this method as a fast and efficient sequencing tool.
A more recently developed sequencing strategy involves sequencing by hybridization on oligonucleotide microchips, or matrices, (SHOM) whereby DNA is hybridized with a complete set of oligonucleotides, which are first immobilized at fixed positions on a glass plate or polyacrylamide gel matrix. There are drawbacks to this technique, however. For instance, given that short nucleotide sequences are repeated rather frequently in long DNA molecules, the sequencing of lengthy genome strings is not feasible via SHOM. Also, hybridization with short oligonucleotides is affected by hairpin structures in DNA.
Furthermore, SHOM requires the utilization of high volume substrates containing many thousands of cells. If immobilized octamers are utilized to determine the positions of each of the four bases in genomic DNA, for example, then 48 or 65,536 such octamers, themselves which would need to be previously fabricated, would have to be immobilized in individual cells on the gel matrix.
The production of literally thousands of these cells on the polyacrylamide substrates is problematic. First, these cells must be accurately spaced relative to one another. Second, these cells must be of sufficient depth and volume to hold predetermined amounts of the oligonucleotide. Cell sizes can range from 25 microns (xcexcm) to 1000 xcexcm.
Typically, cells are produced in a myriad of ways. Two-dimensional scribing techniques and laser evaporation are two typical methods of cell formation. Mechanical scribing techniques are limited, however, in that the smallest structures which can be produced via this method are approximately 100 xcexcmxc3x97100 xcexcm. Lasers applications, because of their expense, also are limiting. Furthermore, both of these procedures require complex equipment and experienced personnel.
A need exists in the art to provide a rapid and efficient method for detecting the existence of complementary sequences to target DNA strands. This detection method should be performed using standard reagents found in a typical biochemistry facility. A need also exists for a method to produce accurate polyacrylamide matrices to be used in the above-disclosed duplex detection method. Such a matrix production method also must be simple enough to be performed in typically-equipped biochemical laboratories.
It is an object of the present invention to provide a method for rapidly detecting the formation and existence of duplexes between complementary nucleotide sequence strands that overcomes many of the disadvantages and reliability shortcomings of the prior art.
Another object of the present invention is to provide a method for the detection of DNA duplexes. A feature of the invention is the use of intercalating dyes. An advantage of the invention is the rapid detection of duplexes using typically-outfitted laboratories to perform standard procedures with common reagents.
Yet another object of the present invention is to provide a highly efficient method for detecting DNA duplexes. A feature of the invention is contacting a DNA duplex, contained on a high-volume support substrate, with an intercalating agent. An advantage of the invention is the enhanced ability to detect small amounts of formed DNA duplexes using standard, low-cost laboratory reagents.
Still another object of the present invention is to provide a method for producing a polyacrylamide matrix having thousands of individual and well defined holding cells. A feature of the invention is the use of mask-controlled photo-polymerization processes. An advantage of the invention is the rendering of high numbers of precise cell geometries and at high densities.
Another object of the present invention is to provide an enhanced method for making arrays to contain oligonucleotide molecules. A feature of the invention is the use of a photosensitive mask with a means for confining reaction fluid, which comprises the array elements, to a light-exposed region during fluid photopolymerization. An advantage of the invention is that the confining means serves to maintain fluid concentration across the array pattern, thereby assuring equal curing and therefore consistency of all of the array elements.
Yet another object of the present invention is to provide a highly sensitive oligonucleotide display array. A feature of the invention is enabling a photopolymerization process of array elements using colorless radical photoinitiators. The photoinitiators have strong absorption maxima at between 180 to 280 nanometers (nm) and are able to produce radicals upon UV radiation. The fluorescence of these photoinitiators are negligible at 400 to 800 nm. An advantage of the invention is the decrease in background fluorescence from gel element constituents and therefore an increase in sensitivity during duplex detection analysis.
Still another object of the present invention is to provide a method for standardizing sequencing and hybridization processes on micro matrices. A feature of the invention is the use of strictly-controlled oligo transfer means. Another feature is comparing the fluorescence intensity of labeled oligomer placed at predetermined positions on the micro matrices to other regions of the micro matrices contacted with labeled, mobile oligomers. An advantage of the invention is the ability to reproduce the hybridization patterns and to determine the efficiency of the hybridizations.
Briefly, the invention provides for a method for determining the existence of duplexes of oligonucleotide complementary molecules comprising constructing a plurality of different oligonucleotide molecules each of a specific length and each having a specific base sequence; supplying a matrix having a plurality of cells adapted to receive and immobilize the oligonucleotide molecules; immobilizing the different oligonucleotide molecules in the cells to fill the cells; contacting the now-filled cells with single stranded oligonucleotide molecules to form a duplex; contacting the duplex with an intercalating agent; and observing fluorescence levels emanating from the now-contacted duplex.
The invention also provides for a method for constructing oligonucleotide matrices comprising confining light sensitive fluid to a surface, exposing said light-sensitive fluid to a light pattern so as to cause the fluid exposed to the light to coalesce into discrete units and stick to the surface; and contacting each of the units with a set of different oligonucleotide molecules so as to allow the molecules to disperse into the units.
The invention further provides for a method for constructing oligonucleotide matrices comprising confining light-sensitive monomer solution to a surface, expo-sing said light-sensitive monomer to a predetermined light pattern so as to cause monomer in solution exposed to the light to polymerize into uniform, discrete gel pads and stick to the surface; and contacting each of the units with a set of different oligonucleotide molecules so as to allow the molecules to disperse into the units